CN103898074A - Glutathione peroxidase GPX1 mutant containing more than two catalysis groups and preparation method thereof - Google Patents

Abstract

The invention discloses a glutathione peroxidase GPX1 mutant containing more than two catalysis groups and a preparation method of the mutant, which belong to the technical filed of biologics. The preparation method comprises the following steps: firstly, acquiring a mutant gene through gene mutation or a synthesis method, secondly, introducing two or more catalysis groups SeCys of GPX into a substrate binding part of the mutant through an auxotroph prokaryotic expression system or an auxotroph and SPP (Single Protein Production) low temperature united expression system, so as to endow the mutant high GPX activity, or firstly, assembling a target gene together with a SeCys insertion sequence into a secreting type mammalia cellular expression carrier, and secondly, assembling SeCys insertion sequence binding protein 2 into an intracellular type mammalia cellular expression carrier, performing cotransfection of the two carriers to one same lactation cell strain, and synthesizing GPX from lactation cells in the presence of sodium selenite. The preparation method is simple, the mutant is high in activity and yield and good in stability, yield decrease and inactivation caused by inclusion body renaturation are avoided, and thus the problems that natural GPX is limited in resource and instable in property are solved.

Description

A kind of Selenoperoxidase GPX1 mutant that contains more than two catalytic group and preparation method thereof

Present patent application is the divisional application of Chinese patent " high vigor Selenoperoxidase GPX1 mutant and preparation method thereof ", the applying date of original application is: 2013-07-18, the application number of original application is: 201310302778.3, and the publication No. of original application is: CN103320406A.

Technical field

The invention belongs to biological technical field, be specifically related to a kind of Selenoperoxidase GPX1 mutant that contains more than two catalytic group and preparation method thereof.

Background technology

The substrate that contains the Selenoperoxidase (GPX) of selenium is gsh (GSH), and catalytic group is seleno-cysteine (SeCys).In vivo, the same superoxide-dismutase of GPX (SOD), catalase (CAT) have formed body anti-oxidative defense system together.GPX plays an important role in this system, it is taking substrate GSH as reductive agent, hydrogen peroxide in decomposer and all kinds of hydroperoxide, thereby can remove activity in vivo oxygen (ROS), prevent lipid peroxidation, the various diseases that treatment is caused by active oxygen, as aging, ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataract, tumour etc.Different from other antioxidase, GPX is except removing ROS, and the lipid peroxide of can also degrading, prevents cell peroxide injury, and the function of the Cell protection of this uniqueness makes it in antioxidase system, occupy the position of particularly important.But, because the source of natural GPX is quite limited, poor stability, cause its artifact and the research of stand-in thereof to receive much concern.

Small molecule mimetics mainly contains PZ51(ebselen), AL3823A, BXT series product, their weakness is that vigor is low, is only the thousandth left and right of natural GPX.The GPX analogue enztme that macromolecular stand-in mainly contain abzyme (Chinese patent 94102481.4 and 96112628.0) and prepare taking glutathione S-transferase (GST) as albumen template, its vigor is significantly higher than small molecule mimetics.Obviously, receive better effect to there is the macromole GPX analogue enztme that the albumen of gsh (GSH) combining site prepared as template, therefore the GPX analogue enztme technology of preparing of these high vigor of exploitation preparation, with regard to the study hotspot of Cheng Liao academia, has broad application prospects for fields such as molecular biology, physiotechnology and medical science.The method that success has been developed so far has: the catalytic group seleno-cysteine (SeCys) of introducing GPX with chemical method (Chinese patent 94102481.4,96112628.0) or auxotroph prokaryotic expression system (Chinese patent 200810050556.6) in non-GPX class template albumen.

Chinese patent 94102481.4,96112628.0,99104234.4, the preparation method of 200810050556.6 disclosed all kinds of selenium-containing abzymes is exactly the catalytic group SeCys that applied chemistry sudden change (modification) method is introduced GPX in antibody class template albumen, there is following shortcoming: (1) is in preparation process, only this step of chemical mutation (modification) will be lost the zymoprotein of 20-40%, causes analogue enztme productive rate significantly to decline; (2) prepare cycle of analogue enztme long, complex operation, the time is long; (3) in chemical mutation process, need to use phenylmethylsulfonyl fluoride, acetonitrile etc., these materials are virose material; (4) lack targeting, for large protein molecular, chemical reaction different loci of being everlasting is introduced multiple non-specific catalytic groups, and therefore chemical mutation is introduced catalytic group and cannot be reached the such specificity of transgenation method.

The another kind of preparation method that Chinese patent 200810050556.6 also discloses people source strand selenium-containing abzyme introduces catalytic group by auxotroph prokaryotic expression system (intestinal bacteria) and transgenation method, but it only limits to the preparation of people source strand selenium-containing abzyme, do not comprise the preparation method of Selenoperoxidase (GPX) mutant and other GPX analogue enztme.Have the glutathione S-transferase (GST) of GPX activity preparation method (Yu, H.J., Liu, J.Q.,

a., Li, J., Luo, G.M., and Shen, J.C.J.Biol.Chem.2005,280,11930-11935) also appear in the newspapers, although this method also adopts auxotroph prokaryotic expression system and transgenation method to introduce catalytic group, it only limits to prepare GPX analogue enztme taking GST as template albumen, does not comprise and prepares GPX mutant taking natural GPX as template.In non-GPX class template albumen, introduce catalytic group to prepare the method for analogue enztme be catalytic group to be incorporated into its that have a same substrate GSH combining site with GPX to plant in albumen and make it produce GPX vigor with auxotroph prokaryotic expression system.

But because these template albumen do not possess the catalytic group of natural GPX self, be therefore difficult to find ideal and the position of catalytic group accurately; Simultaneously, owing to not resembling desirable catalysis triplet natural GPX in these template albumen, therefore the catalytic efficiency of this analoglike enzyme is not high yet.If prepare GPX mutant taking natural GPX as template by gene engineering method, can overcome these shortcomings.Because the codon UGA of catalytic group SeCys of coding GPX is terminator codon, in common prokaryotic expression system, need be in the open reading frame of GPX gene, the codon UGA downstream of next-door neighbour SeCys introduces neck ring structure and UGA could be translated into SeCys instead of terminator codon, and the introducing of neck ring will inevitably cause the change of GPX space conformation in open reading frame, and then affect enzymic activity.Therefore common prokaryotic expression system is unsuitable for directly expressing the seleno-protein that contains with GPX activity.

Summary of the invention

The object of the present invention is to provide Selenoperoxidase GPX1 mutant of a kind of more than two catalytic group and preparation method thereof.

Using gene engineering technique, cell culture technology, auxotroph prokaryotic expression technology and SPP(Single protein production, single protein production) preparation of system hypothermia expression technology has the GPX1 mutant of high GPX vigor.It is the method for directly expressing the GPX1 mutant protein with enzymatic activity high with genetic engineering technique in mammalian cell strain or auxotroph prokaryotic expression system and SPP low temperature expression system thereof.Present method does not need chemically modified to prepare to have the novel artificial enzyme of high GPX vigor.The inventive method has broad application prospects aspect bio-pharmaceuticals.

The present invention with people GPX1(referring to NCBI, NM_000581.2) be template, fit rite-directed mutagenesis by computer mould, obtain a kind of novel high vigor Selenoperoxidase GPX1 mutant, it is made up of 203 amino acid, compare with people GPX1, it does not contain halfcystine, there is higher GPX vigor and better stability, it is that all cysteine mutation in GPX1 are become to Serine, but the 49th remains seleno-cysteine (SeCys, single-letter is abbreviated as U, is write as Xaa in sequence table).The aminoacid sequence (SEQ ID No:1) of described GPX1 mutant is as follows:

MSAARLAAAAAAAQSVYAFSARPLAGGEPVSLGSLRGKVLLIENVASLUGTTVRDYTQMN?ELQRRLGPRGLVVLGFPSNQFGHQENAKNEEILNSLKYVRPGGGFEPNFMLFEKSEVNGAGAHP?LFAFLREALPAPSDDATALMTDPKLITWSPVSRNDVAWNFEKFLVGPDGVPLRRYSRRFQTIDIEP?DIEALLSQGPSSA

Further, the concrete aminoacid sequence of described GPX1 mutant also comprises, any one or more L-Ala (Ala) in above-mentioned aminoacid sequence are sported to any one novel GPX1 mutant that Serine (Ser) forms.Such as 87 L-Ala (Ala) are sported to Serine (Ser), the sequence forming is SEQ ID No:2(embodiment 9).

Further, the concrete aminoacid sequence of described GPX1 mutant also comprises, by any one or more any one novel GPX1 mutant being formed afterwards by seleno-cysteine replacement of the 2nd, 78,115,156,202 5 Serines in above-mentioned aminoacid sequence.Such as the 2nd Serine replaced the rear sequence SEQ ID No:3(embodiment 10 being formed by seleno-cysteine (being write as Xaa in sequence table)).

Further, the concrete aminoacid sequence of described GPX1 mutant also comprises, owing to having used the pColdI(TAKARA company of being convenient to target protein purifying) etc. secretor type protokaryon or carrier for expression of eukaryon and introduce any one novel GPX1 mutant that Histidine purification tag and other amino acid etc. on this carrier form at aminoterminal or the carboxyl terminal of above-mentioned aminoacid sequence.Such as aminoterminal at sequence SEQ ID No:1, SEQ ID No:2 and SEQ ID No:3 is introduced pColdI(TAKARA company) sequence SEQ ID No:4, the SEQ ID No:5 and the SEQ ID No:6(embodiment 11 that form after the Histidine purification tag of prokaryotic expression carrier and the amino acid of factor Xa cleavage site).

The method of the high vigor Selenoperoxidase of preparation of the present invention GPX1 mutant, specifically comprises following method:

Method 1:

First synthesize target gene and be assembled on secretor type prokaryotic expression carrier or first GPX1 genome is installed on secretor type prokaryotic expression carrier, obtain target gene by transgenation (or amino acid substitution) method, again by auxotroph prokaryotic expression system or by auxotroph protokaryon and SPP low temperature associating expression system, the catalytic group seleno-cysteine (SeCys) of GPX is incorporated into the substrate binding site of GPX1 mutant, thereby on this albumen the substrate binding site of existing GPX, there are again catalytic group and the catalysis triplet of GPX, result is given the activity of its high GPX, just produce the Selenoperoxidase GPX1 mutant of high vigor.Method 2:

Or first the gene of coding GPX1 mutant is assembled on secretor type cells of mamma animals expression vector together with selenocysteine insertion sequence, again by selenocysteine insertion sequence in conjunction with albumen 2(SBP2) be assembled in born of the same parents on type cells of mamma animals expression vector, by two kinds of same cells of mamma animals strains of carrier cotransfection, then GPX1 mutant protein is prepared in the positive cell strain that contains GPX1 mutant and two kinds of genes of SBP2 when obtaining with screening, just produce in vitro the genetically engineered Selenoperoxidase GPX1 mutant of high vigor, thereby solve the natural GPX limited problem of originating.

Concrete preparation process of the present invention:

First method of the present invention is the gene that first can express GPX1 mutant protein of the present invention at biotech firm's DNA synthesizer synthetic; guarantee in gene containing ACA sequence, then combine and prepare GPX1 mutant protein with single protein production system and auxotroph prokaryotic expression system.

1), the structure of expression vector: according to the aminoacid sequence of GPX1 mutant of the present invention, can in auxotrophic strain, express the gene of GPX1 mutant protein with DNA synthesizer synthetic in biotech firm, guarantee that 5 of target gene ' end contains initiator codon (ATG), 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 49 seleno-cysteines (SeCys) of target gene replaces with the codon of halfcystine (Cys) (can be TGC etc.), and full length gene does not contain ACA sequence, concrete gene order can be (referring to NCBI by GPX1 gene, NM_000581.2) in the 2nd, 78, 115, the encoding sequence of the halfcystine of 156 and 202 replaces with the codon of Serine, the codon of the 49th seleno-cysteine replaces with the codon of halfcystine, and according to the degeneracy of codon, do not changing under the prerequisite of aminoacid sequence, ACA sequences all in gene is all replaced with to the encoding gene that non-ACA sequence obtains, also can be that other anyly can express GPX1 mutant protein in auxotrophic strain, and total length does not contain the encoding gene of ACA sequence (due to the degeneracy of codon, same aminoacid sequence can have multiple different encoding gene), , then GPX1 mutant gene is assembled on secretor type prokaryotic expression carrier by specific restriction enzyme site with DNA ligase containing the GPX1 mutant gene of specific restriction enzyme site and secretor type prokaryotic expression carrier (as pCold series etc.) with identical restriction endonuclease cutting two ends, described specific restriction enzyme site can be in the multiple clone site of expression vector, contain and non-existent any one restriction enzyme site in GPX1 mutant gene is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier,

2), the screening of positive transformant and the expression and purification of albumen:

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains GPX1 mutant gene building in step 1), be coated with the nutrient agar plate containing Cys, screening positive strain; To contain pMazF(TAKARA, the Cat#3367 of express nucleic acid restriction endonuclease MazF again) plasmid conversion positive strain, with the M9 solid medium screening positive transformant containing dual anti-property; By positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, through 4-25 DEG C of abduction delivering of IPTG low temperature, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX1 mutant that contains SeCys at the combining site of substrate GSH, zymoprotein is expressed and is secreted into soluble form in the pericentral siphon chamber of thalline, and MazF can be by identifying and cut off single stranded RNA particular sequence ACA, suppress the expression of host protein; First cultivate in a small amount the strain of screening high expression level, then enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, ice bath, by liquid low-temperature centrifugation, remove bacterial sediment, obtain supernatant liquor; With gsh (GSH) affinitive layer purification GPX1 mutant protein, after dialysis freeze-drying, obtain GPX1 mutant protein sterling.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein.

The method is by inducing auxotroph prokaryotic expression system to introduce catalytic group at the substrate binding site of GPX1 mutant under low temperature, thereby produces the GPX1 mutant of high vigor.

Described use gsh (GSH) affinitive layer purification GPX1 mutant protein, is with pH7.5, and 50mmol/L Tris-Cl balance wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

Described by liquid low-temperature centrifugation, can be at 4 DEG C, the centrifugal 15-30min of 8000-12000g.

Second method of the present invention is the GPX1 gene that first increases, then taking it as template, obtain the gene that can express GPX1 mutant protein of the present invention by transgenation method, and in the ACA sequence that ensures to suddenly change in gene under the prerequisite that its aminoacid sequence is constant, obtain the not GPX1 mutant gene containing ACA, then combine and prepare GPX1 mutant protein with single protein production system and auxotroph prokaryotic expression system.

1), the structure of expression vector:

According to the gene order of disclosed GPX1 in gene library (referring to NCBI, NM_000581.2) design primer, its encoding gene increases, in the time of design primer, guarantee that 5 of gene ' end contains initiator codon (ATG), 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee the 2nd and the codon of 202 Cys replace to the codon of Ser, other aminoacid sequence is constant; With after identical restriction endonuclease cut vector and target gene, then by specific restriction enzyme site, GPX1 genome is installed to (as pCold series etc.) on secretor type prokaryotic expression carrier with DNA ligase; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport 49 seleno-cysteines of halfcystine and the complete isometric complementation of amino acid whose gene order design near it in GPX1, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the seleno-cysteine in the GPX1 gene being structured on prokaryotic expression carrier is become to the codon of halfcystine; In like manner, guaranteeing not introduce under the prerequisite of ACA sequence by the method for above-mentioned rite-directed mutagenesis, the sequence encoding mutant of other halfcystine in GPX1 gene is become to the codon of Serine, again according to the degeneracy of codon, do not changing under the prerequisite of aminoacid sequence, all ACA series jumps in GPX1 gene are being fallen; Determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee that the gene after sudden change can be expressed GPX1 mutant protein of the present invention in auxotrophic strain; Described specific restriction enzyme site can be in the multiple clone site of expression vector, contain and non-existent any one restriction enzyme site in target gene is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier;

2) screening of positive transformant and the expression and purification of mutant protein

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains GPX1 mutant gene building in step 1), be coated with the nutrient agar plate containing halfcystine, screening positive strain, again the plasmid pMazF of express nucleic acid restriction endonuclease MazF is transformed to positive strain, with the M9 solid medium screening positive transformant containing dual anti-property, by positive transformant spread cultivation support after, containing seleno-cysteine, in the substratum of essential growth factor and nutrient substance, through 4-25 DEG C of abduction delivering of isopropylthio-β-D-galactoside low temperature, auxotrophic strain-BL21 (DE3) Cys can synthesize seleno-cysteine with the codon of halfcystine, directly give expression to the restructuring GPX1 mutant that contains seleno-cysteine at the combining site of substrate gsh, zymoprotein is expressed and is secreted into soluble form in the pericentral siphon chamber of thalline, and MazF can be by identifying and cut off single stranded RNA particular sequence ACA, suppress the expression of host protein, first cultivate in a small amount the strain of screening high expression level, then enlarged culturing and abduction delivering, ultrasonication thalline, release zymoprotein under collection, washing, ice bath, by liquid low-temperature centrifugation, remove bacterial sediment, obtain the supernatant liquor containing GPX1 mutant, with gsh (GSH) affinitive layer purification GPX, after dialysis freeze-drying, obtain zymoprotein sterling.

The method has been introduced catalytic group by inducing auxotroph prokaryotic expression system to combine the single protein production system of SPP under low temperature at the substrate binding site of GPX1 mutant, thereby produces the GPX1 mutant protein of high vigor.

Described use gsh (GSH) affinitive layer purification GPX1 mutant, is with pH7.5, and 50mmol/L Tris-Cl balance wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

Described by liquid low-temperature centrifugation, can be at 4 DEG C, the centrifugal 15-30min of 8000-12000g.

The third method of the present invention is first can express GPX1 mutant protein gene of the present invention at biotech firm's DNA synthesizer synthetic, then prepares GPX1 mutant protein with auxotroph prokaryotic expression system.

1), the structure of expression vector:

According to the aminoacid sequence of GPX1 mutant of the present invention, can in auxotrophic strain, express the gene of GPX1 mutant protein with DNA synthesizer synthetic in biotech firm, guarantee that 5 of target gene ' end contains initiator codon (ATG), 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 49 seleno-cysteines (SeCys) of target gene replaces with the codon of halfcystine (Cys) (can be TGC etc.); Concrete gene order can be (referring to NCBI by GPX1 gene, NM_000581.2) in, the encoding sequence of the halfcystine of the 2nd, 78,115,156 and 202 replaces with the codon of Serine, the codon of the 49th seleno-cysteine replaces with the encoding gene that the codon of halfcystine obtains, also can be other any encoding gene (due to the degeneracy of codon, same aminoacid sequence can have multiple different encoding gene) that can express GPX1 mutant protein in auxotrophic strain; , then GPX1 mutant gene is assembled on secretor type prokaryotic expression carrier by specific restriction enzyme site with DNA ligase containing the GPX1 mutant gene of specific restriction enzyme site and secretor type prokaryotic expression carrier (as pCold series etc.) with identical restriction endonuclease cutting two ends; Described specific restriction enzyme site can be in the multiple clone site of expression vector, to contain and non-existent any one restriction enzyme site in GPX1 mutant gene, is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier;

2), the screening of positive transformant and the expression and purification of albumen:

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains GPX1 mutant gene building in step 1), be coated with the nutrient agar plate containing Cys, screening positive transformant; By positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, induce through isopropylthio-β-D-galactoside (IPTG), auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, finally directly give expression to the GPX1 mutant protein that contains SeCys at the combining site of substrate GSH, zymoprotein is expressed and is secreted into soluble form in the pericentral siphon chamber of thalline; First cultivate in a small amount the strain of screening high expression level, then enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, ice bath, by liquid low-temperature centrifugation, remove bacterial sediment, obtain supernatant liquor; With gsh (GSH) affinitive layer purification GPX1 mutant protein, after dialysis freeze-drying, obtain GPX1 mutant protein sterling.

The method has been introduced catalytic group by transgenation and auxotroph prokaryotic expression system at the substrate binding site of GPX1 mutant, thereby produces the GPX1 mutant protein of high vigor.

Described use gsh (GSH) affinitive layer purification GPX1 mutant protein, is with pH7.5, and 50mmol/L Tris-Cl balance wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

Described by liquid low-temperature centrifugation, can be at 4 DEG C, the centrifugal 15-30min of 8000-12000g.

The 4th kind of method of the present invention is the GPX1 gene that first increases, and then taking it as template, obtains the gene that can express GPX1 mutant protein of the present invention by transgenation method, then prepares GPX1 mutant protein with auxotroph prokaryotic expression system.

1), the structure of expression vector:

According to the gene order of disclosed GPX1 in gene library (referring to NCBI, NM_000581.2) design primer, its encoding gene increases, in the time of design primer, guarantee that 5 of gene ' end contains initiator codon (ATG), 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee the 2nd and the codon of 202 Cys replace to the codon of Ser, other aminoacid sequence is constant; With after identical restriction endonuclease cut vector and target gene, then by specific restriction enzyme site, GPX1 genome is installed to (as pCold series etc.) on secretor type prokaryotic expression carrier with DNA ligase; Keeping under the prerequisite that other aminoacid sequence is constant, according to sporting 49 SeCys of halfcystine (Cys) and two rite-directed mutagenesis primers of its neighbour complete isometric complementation of amino acid whose gene order design in GPX1, centered by the codon of mutating acid, the long 25-50bp of primer; With rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit (Invitrogen company, by the operation of test kit specification sheets), the sequence encoding mutant of the SeCys in the GPX1 gene being structured on prokaryotic expression carrier is become to the codon (such as TGC) of Cys; In like manner, by the method for above-mentioned rite-directed mutagenesis, the sequence encoding mutant of other halfcystine in GPX1 gene is become to the codon of Serine; Determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee that the gene after sudden change can be expressed GPX1 mutant protein of the present invention in auxotrophic strain; Described specific restriction enzyme site can be in the multiple clone site of expression vector, contain and non-existent any one restriction enzyme site in target gene is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier;

2), the screening of positive transformant and the expression and purification of albumen:

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains GPX1 mutant gene building in step 1), be coated with the nutrient agar plate containing Cys, screening positive transformant; By positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, induce through isopropylthio-β-D-galactoside (IPTG), auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, finally directly give expression to the GPX1 mutant protein that contains SeCys at the combining site of substrate GSH, zymoprotein is expressed and is secreted into soluble form in the pericentral siphon chamber of thalline; First cultivate in a small amount the strain of screening high expression level, then enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, ice bath, by liquid low-temperature centrifugation, remove bacterial sediment, obtain supernatant liquor; With gsh (GSH) affinitive layer purification GPX1 mutant protein, after dialysis freeze-drying, obtain GPX1 mutant protein sterling.

The method has been introduced catalytic group by transgenation and auxotroph prokaryotic expression system at the substrate binding site of GPX1 mutant, thereby produces the GPX1 mutant protein of high vigor.

Described use gsh (GSH) affinitive layer purification GPX1 mutant protein, is with pH7.5, and 50mmol/L Tris-Cl balance wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

Described by liquid low-temperature centrifugation, can be at 4 DEG C, the centrifugal 15-30min of 8000-12000g.

Lung biopsy: prepare GPX1 mutant protein with synthetic target gene and mammalian cell expression system

1), the structure of expression vector:

According to the aminoacid sequence of GPX1 mutant of the present invention, encoding gene in biotech firm with DNA synthesizer synthetic GPX1 mutant, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A at whole base sequences of 3 ' end non-translational region of terminator codon downstream introducing GPX1 gene for 3 ' end, concrete sequence is referring to NCBI, NM_000581.2) or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site, concrete gene order can be (referring to NCBI by GPX1 gene, NM_000581.2) in the 2nd, 78, 115, the encoding sequence of the halfcystine of 156 and 202 replaces with the encoding gene that the codon of Serine obtains, also can be that the gene of other any coding GPX1 mutant protein is (due to the degeneracy of codon, same aminoacid sequence can have multiple different encoding gene), but 3 of target gene ' end must contain whole base sequences of 3 of GPX1 gene ' end non-translational region, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A, concrete sequence is referring to NCBI, or selenocysteine insertion sequence (SECIS) NM_000581.2), with identical restriction endonuclease cutting two ends containing the GPX1 mutant gene of specific restriction enzyme site and cells of mamma animals expression vector (as pSecTag2A etc., Invitrogen company), then by specific restriction enzyme site, GPX1 mutant gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence with DNA ligase, according to selenocysteine insertion sequence in gene library in conjunction with albumen 2(SBP2, see NCBI, NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders are at its encoding gene of DNA synthesizer synthetic for biotech firm, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site, SBP2 genome is installed in born of the same parents on type cells of mamma animals expression vector (as pcDNA3.1/myc-His A etc., Invitrogen company) with specific restriction enzyme site, described specific restriction enzyme site can be in the multiple clone site of expression vector, contain and non-existent any one restriction enzyme site in target gene is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier,

2), the screening of positive cell strain:

With the carrier that contains SBP2 and GPX1 mutant gene cotransfection cells of mamma animals under the mediation of transfection reagent, with the resistant gene on two carriers by antibiotic concentration from high to low step by step successive subtraction method screening have the positive cell clone of two kinds of resistances concurrently, with porous culture plate amplification culture positive colony step by step, finally obtain Simultaneous Stabilization and express the positive cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant again; Detect the expression amount of GPX1 mutant protein and screen the cell strain that target protein expression amount is high;

3), the expression and purification of target protein:

In shaking flask, the cell strain amplification culture of two kinds of foreign proteins will be expressed simultaneously, in the substratum that contains Sodium Selenite, express GPX1 mutant with cells of mamma animals, zymoprotein is secreted in substratum with soluble form, with gsh (GSH) affinitive layer purification GPX1 mutant protein, after dialysis freeze-drying, obtain zymoprotein sterling.

The expression amount of described detection GPX1 mutant protein, can detect by the polyclonal antibody of anti-GPX1 and ELISA or GPX vitality test technology;

Described use gsh GSH affinitive layer purification GPX1 mutant protein, is with pH7.5, and 50mmol/L Tris-Cl balance wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

The 6th kind of method of the present invention is the substep carrier that contains SBP2 gene and the same cells of mamma animals of carrier transfection that contains GPX1 mutant gene.

1), the structure of expression vector:

Use the encoding gene of DNA synthesizer synthetic GPX1 mutant in biotech firm according to the aminoacid sequence of GPX1 mutant of the present invention, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A at whole base sequences of 3 ' end non-translational region of terminator codon downstream introducing GPX1 gene for 3 ' end, concrete sequence is referring to NCBI, NM_000581.2) or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site, concrete gene order can be (referring to NCBI by GPX1 gene, NM_000581.2) in the 2nd, 78, 115, the encoding sequence of the halfcystine of 156 and 202 replaces with the encoding gene that the codon of Serine obtains, also can be that the gene of other any coding GPX1 mutant protein is (due to the degeneracy of codon, same aminoacid sequence can have multiple different encoding gene), but 3 of target gene ' end must contain whole base sequences of 3 of GPX1 gene ' end non-translational region, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A, concrete sequence is referring to NCBI, or selenocysteine insertion sequence (SECIS) NM_000581.2), with identical restriction endonuclease cutting two ends containing the GPX1 mutant gene of specific restriction enzyme site and cells of mamma animals expression vector (as pSecTag2A etc., Invitrogen company), then by specific restriction enzyme site, GPX1 mutant gene is assembled on secretor type cells of mamma animals expression vector together with its 3 ' end non-translational region or selenocysteine insertion sequence with DNA ligase, according to selenocysteine insertion sequence in gene library in conjunction with albumen 2(SBP2, see NCBI, NM_024077.3) 512 amino acid whose gene orders of the 343-854 position of carboxyl terminal are at its encoding gene of DNA synthesizer synthetic for biotech firm, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site, SBP2 genome is installed in born of the same parents on type cells of mamma animals expression vector (as pcDNA3.1/myc-His A etc., Invitrogen company) with specific restriction enzyme site, described specific restriction enzyme site can be in the multiple clone site of expression vector, contain and non-existent any one restriction enzyme site in target gene is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier,

2), the screening of positive cell strain:

First with the carrier transfection cells of mamma animals that contains SBP2 gene, by the cell strain of the resistant gene screening stably express SBP2 on this carrier.With the cell strain of the carrier transfection stably express SBP2 that contains GPX1 mutant gene, express the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant with the resistant gene screening Simultaneous Stabilization on two carriers again; Detect the expression amount of GPX1 mutant protein and screen the cell strain that target protein expression amount is high;

3), the expression and purification of target protein:

In shaking flask, will express the cell strain amplification culture of two kinds of foreign proteins simultaneously, in the substratum that contains Sodium Selenite, express GPX1 mutant with cells of mamma animals, zymoprotein is secreted in substratum with soluble form; With gsh affinitive layer purification GPX1 mutant protein, after dialysis freeze-drying, obtain zymoprotein sterling.

In the screening of positive cell strain, the screening of described positive cell strain, also can first transfection GPX1 mutant gene, rear transfection SBP2 gene, then express the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant with the resistant gene screening Simultaneous Stabilization on two carriers.The expression amount of described detection GPX1 mutant protein, can detect by the polyclonal antibody of anti-GPX1 and ELISA or GPX vitality test technology.

Described use gsh affinitive layer purification GPX1 mutant, is with pH7.5, and 50mmol/L Tris-Cl balance wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

The 7th kind of method: with amplification target gene and mammalian cell expression system prepare GPX1 mutant protein

1), the structure of expression vector:

According to GPX1 gene order in gene library (referring to NCBI, NM_000581.2) base sequence of encoding gene and 3 ' end non-translational region of design primer amplification GPX1, in the time of design primer, guarantee that 5 of target gene ' end contains initiator codon (ATG) and specific restriction enzyme site, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A at whole base sequences of 3 ' end non-translational region of terminator codon downstream introducing GPX1 gene for 3 of target gene ' end, concrete sequence is referring to NCBI, or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site NM_000581.2), guarantee that the codon of the 2nd Cys replaces to the codon of Ser, other aminoacid sequence is constant, there are the target gene of specific restriction enzyme site and cells of mamma animals expression vector (as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), then with DNA ligase by specific restriction enzyme site by GPX1 gene together with its 3 ' end non-translational region be assembled on secretor type cells of mamma animals expression vector, keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX1, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the GPX1 gene being structured on carrier for expression of eukaryon is become to the codon of Serine, determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee gene after the sudden change GPX1 mutant protein of the present invention of encoding.According to selenocysteine insertion sequence in gene library in conjunction with albumen 2(SBP2, see NCBI, NM_024077.3) 512 of the 343-854 position of carboxyl terminal amino acid whose its encoding genes of gene order design primer amplification, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site; After cutting connection, enzyme with specific restriction enzyme site, SBP2 genome is installed in born of the same parents on type cells of mamma animals expression vector (as pcDNA3.1/myc-His A etc., Invitrogen company) equally; Described specific restriction enzyme site can be in the multiple clone site of expression vector, contain and non-existent any one restriction enzyme site in target gene is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier;

2), the screening of positive cell strain:

With the carrier that contains SBP2 and GPX1 mutant gene cotransfection cells of mamma animals under the mediation of transfection reagent, with the resistant gene on two carriers by antibiotic concentration from high to low step by step successive subtraction method screening have the positive cell clone of two kinds of resistances concurrently, with porous culture plate amplification culture positive colony step by step, finally obtain Simultaneous Stabilization and express the positive cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant again; Detect the expression amount of GPX1 mutant protein and screen the cell strain that target protein expression amount is high;

3), the expression and purification of target protein:

In shaking flask, the cell strain amplification culture of two kinds of foreign proteins will be expressed simultaneously, in the substratum that contains Sodium Selenite, express GPX1 mutant with cells of mamma animals, zymoprotein is secreted in substratum with soluble form, with gsh (GSH) affinitive layer purification GPX1 mutant protein, after dialysis freeze-drying, obtain zymoprotein sterling.

The expression amount of described detection GPX1 mutant protein, can detect by the polyclonal antibody of anti-GPX1 and ELISA or GPX vitality test technology;

Described use gsh GSH affinitive layer purification GPX1 mutant protein, is with pH7.5, and 50mmol/L Tris-Cl balance wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

The 8th kind of method of the present invention is the substep carrier that contains SBP2 gene and the same cells of mamma animals of carrier transfection that contains GPX1 mutant gene.

1), the structure of expression vector:

According to GPX1 gene order in gene library (referring to NCBI, NM_000581.2) base sequence of encoding gene and 3 ' end non-translational region of design primer amplification GPX1, in the time of design primer, guarantee that 5 of target gene ' end contains initiator codon (ATG) and specific restriction enzyme site, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A at whole base sequences of 3 ' end non-translational region of terminator codon downstream introducing GPX1 gene for 3 of target gene ' end, concrete sequence is referring to NCBI, or selenocysteine insertion sequence (SECIS) and specific restriction enzyme site NM_000581.2), guarantee that the codon of the 2nd Cys replaces to the codon of Ser, other aminoacid sequence is constant, there are the target gene of specific restriction enzyme site and cells of mamma animals expression vector (as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), then with DNA ligase by specific restriction enzyme site by GPX1 gene together with its 3 ' end non-translational region be assembled on secretor type cells of mamma animals expression vector, keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX1, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the GPX1 gene being structured on carrier for expression of eukaryon is become to the codon of Serine, determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee gene after the sudden change GPX1 mutant protein of the present invention of can encoding.According to selenocysteine insertion sequence in gene library in conjunction with albumen 2(SBP2, see NCBI, NM_024077.3) 512 of the 343-854 position of carboxyl terminal amino acid whose its encoding genes of gene order design primer amplification, guarantee that 5 of gene ' end contains initiator codon (ATG) and specific restriction enzyme site, 3 ' end contains terminator codon and specific restriction enzyme site; After cutting connection, enzyme with specific restriction enzyme site, SBP2 genome is installed in born of the same parents on type cells of mamma animals expression vector (as pcDNA3.1/myc-His A etc., Invitrogen company) equally; Described specific restriction enzyme site can be in the multiple clone site of expression vector, contain and non-existent any one restriction enzyme site in target gene is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier;

2), the screening of positive cell strain:

First with the carrier transfection cells of mamma animals that contains SBP2 gene, by the cell strain of the resistant gene screening stably express SBP2 on this carrier; With the cell strain of the carrier transfection stably express SBP2 that contains GPX1 mutant gene, express the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant with the resistant gene screening Simultaneous Stabilization on two carriers again; Detect the expression amount of GPX1 mutant protein and screen the cell strain that target protein expression amount is high;

3), the expression and purification of target protein:

In shaking flask, will express the cell strain amplification culture of two kinds of foreign proteins simultaneously, in the substratum that contains Sodium Selenite, express GPX1 mutant with cells of mamma animals, zymoprotein is secreted in substratum with soluble form; With gsh affinitive layer purification GPX1 mutant protein, after dialysis freeze-drying, obtain zymoprotein sterling.

In the screening of positive cell strain, the screening of described positive cell strain, also can first transfection GPX1 mutant gene, rear transfection SBP2 gene, then express the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant with the resistant gene screening Simultaneous Stabilization on two carriers.The expression amount of described detection GPX1 mutant protein, can detect by the polyclonal antibody of anti-GPX1 and ELISA or GPX vitality test technology.

Described use gsh affinitive layer purification GPX1 mutant, is with pH7.5, and 50mmol/L Tris-Cl balance wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

The 9th kind of method of the present invention is identical with first method, just the encoding sequence of the 87th L-Ala is replaced with when the encoding gene of synthetic GPX1 mutant to the codon of Serine, the 87th of the GPX1 mutant protein finally obtaining is Serine (SEQ ID No:2), instead of L-Ala, other aminoacid sequence is identical with first method.

The of the present invention ten kind of method is identical with first method, just the encoding sequence of second Serine is replaced with when the encoding gene of synthetic GPX1 mutant to the codon of halfcystine, the second of the GPX1 mutant protein finally obtaining is seleno-cysteine (SEQ ID No:3), instead of Serine, other aminoacid sequence is identical with first method.

The 11 kind of method of the present invention is identical with first method, just owing to having used the pColdI(TAKARA company of being convenient to target protein purifying) etc. secretor type protokaryon or carrier for expression of eukaryon and introduce any one novel GPX1 mutant that Histidine purification tag and other amino acid etc. on this carrier form at aminoterminal or the carboxyl terminal of target protein.Such as aminoterminal at sequence SEQ ID No:1, SEQ ID No:2, SEQ ID No:3 is introduced pColdI(TAKARA company) sequence SEQ ID No:4, the SEQ ID No:5, the SEQ ID No:6 that form after the Histidine purification tag of prokaryotic expression carrier and the amino acid of factor Xa cleavage site.The present invention has following characteristics:

(1) the present invention uses the genetic engineering techniques such as simple gene synthetic or amplification, transgenation and expression, and preparation method is simple.

(2) the GPX1 mutant protein vigor that prepared by the inventive method is high, has exceeded an order of magnitude of natural GPX vigor, and Cell Biology Experiment has confirmed that the myocardial cell of Hydroperoxide injury is had to stronger provide protection.

(3) GPX1 mutant protein of the present invention has superpower stability, is difficult for inactivation, and this characteristic has made up the defect of natural GPX.

(4) the secreted expression carrier that the present invention uses, GPX1 mutant protein can be expressed and is secreted into the external of colibacillary pericentral siphon chamber or cells of mamma animals with soluble form, the targeting signal peptide of pilot protein secreting, expressing is excised automatically by thalline or cell, expressed albumen is solubility, there is the activity of space conformation and the Geng Gao of native protein, do not need renaturation, can avoid the productive rate that renaturing inclusion bodies process causes to decline and inactivation, thus with short production cycle.

(5) the characteristic that the SPP low temperature expression system that the present invention uses can utilize MazF proteolytic enzyme specific recognition and cut the mRNA that contains ACA sequence, make thalline can only composite coding gene in without the protein of ACA sequence, therefore new synthetic albumen is mainly foreign protein, thereby improve productive rate and Cys to the transformation efficiency of SeCys, therefore there is the double dominant that vigor is high, productive rate is high.

(6) the present invention's eukaryotic expression used directly uses the selenocysteine insertion sequence (SECIS) of GPX self, do not need to introduce in addition SECIS and enter expression vector, thereby simple to operate and available conventional carrier for expression of eukaryon is implemented.

These advantages are all conducive to scale operation, for practical application from now on lays a solid foundation, have solved the problem that natural GPX source is not enough, character is unstable and vigor is low, aspect bio-pharmaceuticals, have broad application prospects.

Figure of description

Fig. 1: on the SDS-PAGE(of the GPX1 mutant protein that purifying obtains) and Western blot(under) result: wherein M is molecular weight of albumen Marker, and 1-11 swimming lane is the target protein of embodiment 1～11 preparation.

Embodiment

Embodiment 1: prepare genetically engineered people GPX1 mutant protein with synthetic gene associating SPP and auxotroph prokaryotic expression system

The sequence 1(SEQ ID No:1 according to the present invention) aminoacid sequence of described GPX1 mutant, biotech firm with DNA synthesizer synthetic can be in auxotrophic strain expressed sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, guarantee that 5 of GPX1 mutant gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, the encoding sequence TGA of No. 49 SeCys of GPX1 mutant gene replaces with the codon (TGC) of Cys, and full length gene does not contain ACA sequence, concrete target-gene sequence is (referring to NCBI by GPX1 gene, NM_000581.2) in the 2nd, 78, 115, the encoding sequence that the codon of the halfcystine of 156 and 202 replaces with Serine (successively: AGT, AGC, AGC, AGT, AGT), the codon of the 49th seleno-cysteine replaces with the encoding sequence (TGC) of halfcystine, and by gene the 168th, 537, 561 and No. 573 base C all replaces with the GPX1 mutant gene that does not contain ACA sequence that T obtains, guarantee this gene energy code book invention sequence 1(SEQ ID No:1) described GPX1 mutant protein, and 5 of target gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, with after restriction endonuclease Nde I and Hind III double digestion, be connected to pCold III(TAKARA, Cat.#3369 that same enzyme is cut) on carrier.

With carrier (pCold III-GPX1 is prominent) conversion auxotroph e. coli bl21 (DE3) Cys that GPX1 mutant gene is housed, be coated with the nutrient agar plate that contains Cys, screening positive strain.Again by plasmid pMazF(TAKARA, the Cat.#3369 of express nucleic acid restriction endonuclease MazF) be transformed in this positive strain.Bacterium liquid is uniformly coated in the M9-CAA solid medium containing 100 μ g/mL Amp, 25 μ g/mL Kana and 25 μ g/mL and screens positive transformant.By positive transformant be inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and the each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.5.Bacterium liquid is placed in to-20 DEG C of refrigerator 5min, makes bacterium liquid cooling rapidly, afterwards bacterium liquid is placed in to 15 DEG C of relaying persistent oscillations and cultivates 45min.15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant.With the resuspended bacterial sediment of 0.9%NaCl, 15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant, repeat this step.Then use the resuspended thalline of productive culture base (adding the each 50-400 μ of the 19 seed amino acid g/ml except Cys in M9 substratum), adding final concentration is that 1mmol/L IPTG and final concentration are 200 μ g/mL SeCys, 15 DEG C of shaking culture 16-72h, GPX1 mutant protein is expressed in the pericentral siphon chamber of thalline with soluble form, and MazF can, by identifying and cut off single stranded RNA particular sequence ACA, suppress the expression of host protein.Collect thalline (6000rpm, 10min) also with isopyknic bufferT(50mM Tris buffer, pH7.5, containing 1mM EDTA) washed twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collects supernatant.By specification is processed GSH affinity post, and application damping fluid (50mmol/L Tris, pH7.5) balance, adds supernatant liquor on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein.By elutriant dialysis freeze-drying, obtain people GPX1 mutant protein.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein, its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 1st swimming lane of Fig. 1.Its GPX vigor is 21786U/ μ mol, exceedes order of magnitude of natural GPX.

The characteristic that the SPP low temperature expression system that the present embodiment uses can utilize MazF proteolytic enzyme specific recognition and cut the mRNA that contains ACA sequence, make thalline can only composite coding gene in without the protein of ACA sequence, therefore new synthetic albumen is mainly external source recombinant protein, thereby improve productive rate and the Cys transformation efficiency to SeCys, therefore there is the high high yield double dominant of living.

Embodiment 2: prepare genetically engineered people GPX1 mutant protein with gene amplification and sudden change method and SPP associating auxotroph prokaryotic expression system

Extract in a small amount test kit (Sigma with mRNA, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, extract mRNA, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist under, be transcribed into cDNA by RT-PCR (RT-polymerase chain reaction).According to the gene of disclosed people GPX1 in gene library (referring to NCBI, NM_000581.2) primers its encoding gene that increases, guarantee that 5 of gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, and by the 2nd and the codon of No. 202 Cys replace to the codon of Ser, other aminoacid sequence is constant; Concrete 5 ' end primer is 5 '-GGAATTCCATATGAGTGCTGCTCGGC-3 ', and 3 ' end primer is 5 '-CCCAAGCTTCTAGGCACTGCTGGGC-3 '.With after restriction endonuclease Nde I and Hind III double digestion, be connected to pCold III(TAKARA, the Cat.#3369 that same enzyme is cut with DNA ligase) on carrier.Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers of the complete isometric complementation of amino acid whose gene order design near No. 49 SeCys, the long 25-50bp of primer, centered by the codon (TGA) of No. 49 SeCys, the sequence of sense primer is 5 '-GAATGTGGCGTCCCTCTGCGGCACCACGGTCCGGGAC-3 '.With primer and Fast Fixed-point sudden change test kit (the Invitrogen company of these two complete complementaries, press the operation of test kit specification sheets), the encoding sequence TGA of No. 49 SeCys that is structured in the GPX1 gene on prokaryotic expression carrier pCold III is mutated into the codon (TGC) of Cys, determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation.With same method, the codon mutation of other all Cys in people GPX1 gene is become to the codon of Ser; Corresponding sense primer is respectively: C78S5 '-GTGCTCGGCTTCCCGAGCAACCAGTTTGGGC-3 ', C115S5 '-CATGCTCTTCGAGAAGAGCGAGGTGAACGGTGC-3 ', C156S5 '-CACCTGGTCTCCGGTGAGTCGCAACGATGTTGC-3 '.Finally keep under the prerequisite that GPX1 mutant aminoacid sequence is constant sudden change fall gene in all ACA sequences, be about to the 168th, 537,561 and No. 573 base C and all sport T, share 3 primers, its just sequence is respectively 5 '-CACGGTCCGGGACTATACCCAGATGAACGAG-3 ', 5 '-CCCCTACGCAGGTATAGCCGCCGCTTCC-3 ', 5 '-CGCTTCCAGACCATTGATATCGAGCCTGATATCGAAGCCCTGCTGTC-3 '; Determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene energy code book invention sequence 1(Seq1 after sudden change) described GPX1 mutant protein.

With carrier (pCold III-GPX1 is prominent) conversion auxotroph e. coli bl21 (DE3) Cys that GPX1 mutant gene is housed, be coated with the nutrient agar plate that contains Cys, screening positive strain.Again by plasmid pMazF(TAKARA, the Cat.#3369 of express nucleic acid restriction endonuclease MazF) be transformed in this positive strain.Bacterium liquid is uniformly coated in the M9-CAA solid medium containing 100 μ g/mL Amp, 25 μ g/mL Kana and 25 μ g/mL and screens positive transformant.By positive transformant be inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and the each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.5.Bacterium liquid is placed in to-20 DEG C of refrigerator 5min, makes bacterium liquid cooling rapidly, afterwards bacterium liquid is placed in to 15 DEG C of relaying persistent oscillations and cultivates 45min.15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant.The resuspended bacterial sediment of 0.9%NaCl, 15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant, repeat this step.Then use the resuspended thalline of productive culture base (adding the each 50-400 μ of the 19 seed amino acid g/ml except Cys in M9 substratum), adding final concentration is that 1mmol/L IPTG and final concentration are 200 μ g/mL SeCys, 15 DEG C of shaking culture 16-72h, GPX1 mutant protein is expressed in the pericentral siphon chamber of thalline with soluble form, and MazF can, by identifying and cut off single stranded RNA particular sequence ACA, suppress the expression of host protein.Collect thalline (6000rpm, 10min) also with isopyknic bufferT(50mM Tris buffer, pH7.5, containing 1mM EDTA) washed twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collects supernatant.By specification is processed GSH affinity post, and application damping fluid (50mmol/L Tris, pH7.5) balance, adds supernatant liquor on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein.By elutriant dialysis freeze-drying, obtain people GPX1 mutant protein.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein, its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 2nd swimming lane of Fig. 1.Its GPX vigor is 21560U/ μ mol, exceedes order of magnitude of natural GPX.

The characteristic that the SPP low temperature expression system that the present embodiment uses can utilize MazF proteolytic enzyme specific recognition and cut the mRNA that contains ACA sequence, make thalline can only composite coding gene in without the protein of ACA sequence, therefore new synthetic albumen is mainly external source recombinant protein, thereby improve productive rate and the Cys transformation efficiency to SeCys, therefore there is the high high yield double dominant of living.

Embodiment 3: prepare genetically engineered people GPX1 mutant protein with synthetic gene and auxotroph prokaryotic expression system

The sequence 1(SEQ ID No:1 according to the present invention) aminoacid sequence of described GPX1 mutant, biotech firm with DNA synthesizer synthetic can be in auxotrophic strain expressed sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, guarantee that 5 of GPX1 mutant gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, and the encoding sequence TGA of No. 49 SeCys of GPX1 mutant gene replaces with the codon (TGC) of Cys, concrete target-gene sequence is (referring to NCBI by GPX1 gene, NM_000581.2) in the 2nd, 78, 115, the encoding sequence that the codon of the halfcystine of 156 and 202 replaces with Serine (successively: AGT, AGC, AGC, AGT, AGT), what the codon of the 49th seleno-cysteine replaced with that the encoding sequence (TGC) of halfcystine obtains can code book invention sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, and 5 of target gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, with after restriction endonuclease Nde I and HindIII double digestion, be connected to pCold III(TAKARA, Cat.#3369 that same enzyme is cut) on carrier.

With carrier (pCold III-GPX1 is prominent) conversion auxotroph e. coli bl21 (DE3) Cys that GPX1 mutant gene is housed, be coated with the nutrient agar plate that contains Cys, screening positive transformant.By positive transformant be inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and the each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.1, be cultured to OD600 and be about 0.3-1, add the IPTG of final concentration 0.1-1mM, after 10min, add the paraxin of final concentration 10 μ g/ml.Add paraxin medium centrifugal after five minutes, centrifugal 5 minutes of low temperature 6000rpm, wash twice with the physiological salt solution of ice bath, each with 1.2L, then use productive culture base (adding the each 50-400 μ of the 19 seed amino acid g/ml except Cys in M9 substratum) resuspended, and to the Rifampin and the 600 μ M SeCys that supplement final concentration 400 μ g in substratum.Continue to cultivate 2-12h, GPX1 is expressed in the pericentral siphon chamber of thalline with soluble form.Collect thalline (6000rpm, 10min) also with isopyknic bufferT(50mM Tris buffer, pH7.5, containing 1mM EDTA) washed twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collects supernatant.By specification is processed GSH affinity post, and application damping fluid (50mmol/L Tris, pH7.5) balance, adds supernatant liquor on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein.By elutriant dialysis freeze-drying, obtain people GPX1 mutant protein.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein, its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 3rd swimming lane of Fig. 1.Its GPX vigor is 5320U/ μ mol, reaches the order of magnitude level of natural GPX.

Auxotroph e. coli bl21 (DE3) Cys derives from the article that exercise question is " Structure of the Cathelicidin Motif of Protegrin-3Precursor:Structural Insights into the Activation Mechanism of an Antimicrobial Protein ", within 2002, be published in Structure the 10th volume, 1,363 1370 pages of –.Obtaining of this bacterial classification can be granted by author or August professor Bock.

Embodiment 4: prepare genetically engineered people GPX1 mutant protein with gene amplification and sudden change method and auxotroph prokaryotic expression system

Extract in a small amount test kit (Sigma with mRNA, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, extract mRNA, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist under, be transcribed into cDNA by RT-PCR (RT-polymerase chain reaction).According to the gene of disclosed people GPX1 in gene library (referring to NCBI, NM_000581.2) primers its encoding gene that increases, guarantee that 5 of gene ' end contains initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end contains terminator codon and Hind III restriction enzyme site, and by the 2nd and the codon of No. 202 Cys replace to the codon of Ser, other aminoacid sequence is constant; Concrete 5 ' end primer is 5 '-GGAATTCCATATGAGTGCTGCTCGGC-3 ', and 3 ' end primer is 5 '-CCCAAGCTTCTAGGCACTGCTGGGC-3 '.With after restriction endonuclease Nde I and Hind III double digestion, be connected to pCold III(TAKARA, the Cat.#3369 that same enzyme is cut with DNA ligase) on carrier.Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers of the complete isometric complementation of amino acid whose gene order design near No. 49 SeCys, the long 25-50bp of primer, centered by the codon (TGA) of No. 49 SeCys.With primer and Fast Fixed-point sudden change test kit (the Invitrogen company of these two complete complementaries, press the operation of test kit specification sheets), the encoding sequence TGA of No. 49 SeCys that is structured in the GPX1 gene on prokaryotic expression carrier pCold III is mutated into the codon (TGC) of Cys, determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, with same method, the sequence encoding mutant of other all Cys in people GPX1 gene is become to the codon of Ser, the codon that is Ser by the sequence encoding mutant of 87 Ala (TCC), corresponding sense primer is respectively: C78S5 '-GTGCTCGGCTTCCCGAGCAACCAGTTTGGGC-3 ', C115S5 '-CATGCTCTTCGAGAAGAGCGAGGTGAACGGTGC-3 ', C156S5 '-CACCTGGTCTCCGGTGAGTCGCAACGATGTTGC-3 ' and A87S5 '-GTTTGGGCATCAGGAGAACTCCAAGAACGAAGAGATTC-3 '.Determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene energy code book invention sequence 2(SEQ ID No:2 after sudden change) described GPX1 mutant protein.

With carrier (pCold III-GPX1 is prominent) conversion auxotroph e. coli bl21 (DE3) Cys that GPX1 mutant gene is housed, be coated with the nutrient agar plate that contains Cys, screening positive transformant.By positive transformant be inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and the each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.1, be cultured to OD600 and be about 0.3-1, add the IPTG of final concentration 0.1-1mM, after 10min, add the paraxin of final concentration 10 μ g/ml.Add paraxin medium centrifugal after five minutes, centrifugal 5 minutes of low temperature 6000rpm, wash twice with the physiological salt solution of ice bath, each with 1.2L, then use productive culture base (adding the each 50-400 μ of the 19 seed amino acid g/ml except Cys in M9 substratum) resuspended, and to the Rifampin and the 600 μ M SeCys that supplement final concentration 400 μ g in substratum.Continue to cultivate 2-12h, GPX1 is expressed in the pericentral siphon chamber of thalline with soluble form.Collect thalline (6000rpm, 10min) also with isopyknic bufferT(50mM Tris buffer, pH7.5, containing 1mM EDTA) washed twice.With the resuspended bacterial sediment of BufferT, add the PMSF(phenylmethylsulfonyl fluoride of final concentration 1mM), ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collects supernatant.By specification is processed GSH affinity post, and application damping fluid (50mmol/L Tris, pH7.5) balance, adds supernatant liquor on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein.By elutriant dialysis freeze-drying, obtain people GPX1 mutant protein.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein, its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 4th swimming lane of Fig. 1.Its GPX vigor is 5130U/ μ mol, reaches the order of magnitude level of natural GPX.The GPX1 mutant of the aminoacid sequence of (present method also can be used for preparing 87 Ala do not sport Serine there is sequence 1(SEQ ID No:1), concrete grammar is except this step of codon that to save the sequence encoding mutant of 87 Ala be Ser, and other is identical with this law.）

Embodiment 5: prepare people GPX1 mutant protein with synthetic gene and the one step transfection of mammalian cell expression system

1. the structure of people GPX1 mutant protein expression vector: the sequence 1(SEQ ID No:1 according to the present invention) aminoacid sequence of described GPX1 mutant is at DNA synthesizer synthetic GPX1 mutant gene for biological reagent company, guarantee that 5 of gene ' end contains initiator codon (ATG) and Hind III restriction enzyme site, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A at whole base sequences of 3 of people GPX1 mutant gene terminator codon downstream GPX1 gene ' end non-translational region for 3 ' end, concrete sequence is referring to NCBI, NM_000581.2), and insert BamHI restriction enzyme site after the previous base of the poly oligonucleotide A of GPX1 gene, concrete target-gene sequence is (referring to NCBI by GPX1 gene, NM_000581.2) in the 2nd, 78, 115, the encoding sequence that the codon of the halfcystine of 156 and 202 replaces with Serine (successively: AGT, AGC, AGC, AGT, AGT) obtain can code book invention sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, and (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A for whole base sequences that 3 of target gene ' end contains 3 of GPX1 gene ' end non-translational region, concrete sequence is referring to NCBI, NM_000581.2), and insert BamHI restriction enzyme site after the previous base of the poly oligonucleotide A of GPX1 gene.Cut latter linked method with first enzyme, by Hind III/BamHI restriction enzyme site, GPX1 mutant gene is connected on the cells of mamma animals expression vector pSecTag2A (Invitrogen company) that same enzyme cuts together with selenocysteine insertion sequence; In like manner according to human selenocysteine insertion sequence in gene library in conjunction with albumen 2(SBP2; NCBI; NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders are at its encoding gene of DNA synthesizer synthetic for biotech firm; guarantee that 5 of gene ' end contains initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end contains terminator codon and EcoRI restriction enzyme site.Cut latter linked method with first enzyme, by XbaI/EcoRI restriction enzyme site, the carboxyl terminal of SBP2 (343-854aa) genome is installed in the born of the same parents that same enzyme cuts on type cells of mamma animals expression vector pcDNA3.1/myc-His A (Invitrogen company).

2. the screening of people GPX1 mutant gene positive cell strain: when Growth of Cells to 1 × 10 ⁶when the density of/ml, with the expression vector that contains SBP2 and GPX1 mutant gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower cotransfection 293F cell (Invitrogen of mediation, R79007), cell was proceeded to and in culture dish, starts adherent culture in after transfection 72 hours, the common 2-50 of adherent culture 48() hour after change the nutrient solution that contains G418 and Zeocin, it is the positive cell clone that Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant that screening has two kinds of resistances, G418 and Zeocin concentration are progressively successively decreased from 800-200 μ g/ml and 400-100 μ g/ml respectively during this time, subtract respectively 200 μ g/ml and 100 μ g/ml at every turn, each concentration is used 5 days, within 2-3 days, change nutrient solution one time, screening and culturing after 20 days by positive cell clone with 96, 48, 24, 12 and 6 well culture plates amplification culture step by step.Detect the expression amount of GPX1 mutant protein with anti-GPX1 polyclonal antibody and elisa technique, select the cell strain that target protein expression amount is high and support with frozen for spreading cultivation.

3. the expression and purification of people GPX1 mutant protein: the Simultaneous Stabilization that during the 293F that contains 1-10 μ M Sodium Selenite at large capacity spreads cultivation in expressing substratum and supports 2, screening obtains is expressed the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant.In SBP2, SECIS and 293 cells, under the common participation of other protein factor, GPX1 mutant protein is expressed with soluble form and is secreted in substratum, within every 48 hours, collects a nutrient solution.Nutrient solution, after concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, with GSH affinity chromatography column purification GPX1 mutant protein, is collected the elutriant that contains GPX4, and small-molecule substance is removed in dialysis, and freeze-drying obtains people GPX1 mutant protein.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein, its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 5th swimming lane of Fig. 1.Its GPX vigor is 22689U/ μ mol, exceedes order of magnitude of natural GPX.

Embodiment 6: prepare people GPX1 mutant protein with the transfection of synthetic gene and mammalian cell expression system substep

1. the structure of people GPX1 mutant protein expression vector: the sequence 1(SEQ ID No:1 according to the present invention) aminoacid sequence of described GPX1 mutant is at DNA synthesizer synthetic GPX1 mutant gene for biological reagent company, guarantee that 5 of gene ' end contains initiator codon (ATG) and Hind III restriction enzyme site, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A at whole base sequences of 3 of people GPX1 mutant gene terminator codon downstream GPX1 gene ' end non-translational region for 3 ' end, concrete sequence is referring to NCBI, NM_000581.2), and insert BamHI restriction enzyme site after the previous base of the poly oligonucleotide A of GPX1 gene, concrete target-gene sequence is (referring to NCBI by GPX1 gene, NM_000581.2) in the 2nd, 78, 115, the encoding sequence that the codon of the halfcystine of 156 and 202 replaces with Serine (successively: AGT, AGC, AGC, AGT, AGT) obtain can code book invention sequence 1(SEQ ID No:1) gene of described GPX1 mutant protein, and (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A for whole base sequences that 3 of target gene ' end contains 3 of GPX1 gene ' end non-translational region, concrete sequence is referring to NCBI, NM_000581.2), and insert BamHI restriction enzyme site after the previous base of the poly oligonucleotide A of GPX1 gene.Cut latter linked method with first enzyme, by Hind III/BamHI restriction enzyme site, GPX1 mutant gene is connected on the cells of mamma animals expression vector pSecTag2A (Invitrogen company) that same enzyme cuts together with selenocysteine insertion sequence; In like manner according to human selenocysteine insertion sequence in gene library in conjunction with albumen 2(SBP2; NCBI; NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders are at its encoding gene of DNA synthesizer synthetic for biotech firm; guarantee that 5 of gene ' end contains initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end contains terminator codon and EcoRI restriction enzyme site.Cut latter linked method with first enzyme, by XbaI/EcoRI restriction enzyme site, the carboxyl terminal of SBP2 (343-854aa) genome is installed in the born of the same parents that same enzyme cuts on type cells of mamma animals expression vector pcDNA3.1/myc-His A (Invitrogen company).

2. the screening of people GPX1 mutant gene positive cell strain: when Growth of Cells to 1 × 10 ⁶when the density of/ml, with the expression vector that contains SBP2 gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower transfection 293F cell (Invitrogen of mediation, R79007), cell was proceeded to and in culture dish, starts adherent culture in after transfection 72 hours, the common 2-50 of adherent culture 48() hour after change the nutrient solution that contains G418, screening has the positive cell clone that G418 resistance is stably express SBP2, G418 concentration is progressively decremented to 200 μ g/ml from 800 μ g/ml during this time, subtract 200 μ g/ml at every turn, each concentration is used 5 days, within 2-3 days, change nutrient solution one time, screening and culturing after 20 days by positive cell clone with 96, 48, 24, 12 and 6 well culture plates amplification culture step by step.Use again the expression vector that contains GPX1 mutant gene at lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the mediation 293F cell (Invitrogen of transfection stably express SBP2 down, R79007), cell was proceeded to and in culture dish, starts adherent culture in after transfection 72 hours, the common 2-50 of adherent culture 48() hour after change the nutrient solution screening that contains G418 and Zeocin to have two kinds of resistances be the positive cell clone that Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant, G418 concentration maintains 200 μ g/ml during this time, Zeocin concentration is progressively decremented to 100 μ g/ml from 400 μ g/ml, subtract 100 μ g/ml at every turn, each concentration is used 5 days, within 2-3 days, change nutrient solution one time, screening and culturing after 20 days by the positive cell clone with two kinds of resistances with 96, 48, 24, 12 and 6 well culture plates amplification culture step by step.With the polyclonal antibody of anti-GPX1 and the expression amount of elisa technique detection GPX1 mutant protein, select the cell strain that target protein expression amount is high and support with frozen for spreading cultivation.

Aforesaid method also can first transfection GPX1 mutant gene, rear transfection SBP2 gene.

3. the expression and purification of people GPX1 mutant: the Simultaneous Stabilization that during the 293F that contains 1-10 μ M Sodium Selenite at large capacity spreads cultivation in expressing substratum and supports 2, screening obtains is expressed the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant.In SBP2, SECIS and 293F cell, under the common participation of other protein factor, GPX1 mutant is expressed with soluble form and is secreted in substratum, within every 48 hours, collects a nutrient solution.Nutrient solution, after concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, with GSH affinity chromatography column purification GPX1 mutant, is collected the elutriant that contains GPX1 mutant, and small-molecule substance is removed in dialysis, and freeze-drying obtains people GPX1 mutant protein.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein, its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 6th swimming lane of Fig. 1.Its GPX vigor is 21968U/ μ mol, exceedes order of magnitude of natural GPX.

Embodiment 7: prepare people GPX1 mutant protein with the target gene of amplification with the one step transfection of mammalian cell expression system

1. the structure of people GPX1 mutant protein expression vector: extract in a small amount test kit (Sigma with mRNA, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, extract mRNA, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist under, be transcribed into cDNA by RT-PCR (RT-polymerase chain reaction).According to the gene order of disclosed people GPX1 in gene library (referring to NCBI, NM_000581.2) base sequence of encoding gene and 3 ' end non-translational region of design primer amplification GPX1, in the time of design primer, guarantee that 5 of target gene ' end contains initiator codon (ATG) and Hind III restriction enzyme site, the codon of the 2nd Cys replaces with the encoding sequence of Ser, other aminoacid sequence is constant, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A at whole base sequences of 3 ' end non-translational region of terminator codon downstream introducing GPX1 gene for 3 ' end, concrete sequence is referring to NCBI, and Hind III restriction enzyme site NM_000581.2), there are the target gene of specific restriction enzyme site and cells of mamma animals expression vector (as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), then with DNA ligase by specific restriction enzyme site by GPX1 gene together with its 3 ' end non-translational region be assembled on secretor type cells of mamma animals expression vector, concrete 5 ' end primer is 5 '-CCCAAGCTTCATATGAGTGCTGCTCGGC-3 ', and 3 ' end primer is 5 '-CGGGATCCAGT GGGGAAACTCG-3 '.Cut latter linked method with first enzyme, by Hind III/BamHI restriction enzyme site, GPX1 gene is connected on the cells of mamma animals expression vector pSecTag2A (Invitrogen company) that same enzyme cuts together with selenocysteine insertion sequence; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX1, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the people GPX1 gene being structured on prokaryotic expression carrier is become to the codon of Serine; Concrete sense primer is respectively: C78S5 '-GTGCTCGGCTTCCCGAGCAACCAGTTTGGGC-3 ', C115S5 '-CATGCTCTTCGAGAAGAGCGAGGTGAACGGTGC-3 ', C156S5 '-CACCTGGTCTCCGGTGAGTCGCAACGATGTTGC-3 ' and C202S5 '-GTCTCAAGGGCCCAGCTCTGCCTAGGGCGCCCCTC-3 '; Determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene energy code book invention sequence 1(SEQ ID No:1 after sudden change) described GPX1 mutant protein.According to human selenocysteine insertion sequence in gene library in conjunction with albumen 2(SBP2, NCBI, NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders, its encoding gene of design primer amplification, guarantee that 5 of gene ' end contains initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end contains terminator codon and EcoRI restriction enzyme site; Concrete 5 ' end primer is 5 '-GCTCTAGAATGGAAGCTTTATCTTC-3 ', and 3 ' end primer is 5 '-CGGAATTCTAAATTCAAATTC-3 '.Cut latter linked method with first enzyme, by XbaI/EcoRI restriction enzyme site, the carboxyl terminal of SBP2 (343-854aa) genome is installed in the born of the same parents that same enzyme cuts on type cells of mamma animals expression vector pcDNA3.1/myc-His A (Invitrogen company).

2. the screening of people GPX1 mutant gene positive cell strain: when Growth of Cells to 1 × 10 ⁶when the density of/ml, with the expression vector that contains SBP2 and GPX1 mutant gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower cotransfection 293F cell (Invitrogen of mediation, R79007), cell was proceeded to and in culture dish, starts adherent culture in after transfection 72 hours, the common 2-50 of adherent culture 48() hour after change the nutrient solution that contains G418 and Zeocin, it is the positive cell clone that Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant that screening has two kinds of resistances, G418 and Zeocin concentration are progressively successively decreased from 800-200 μ g/ml and 400-100 μ g/ml respectively during this time, subtract respectively 200 μ g/ml and 100 μ g/ml at every turn, each concentration is used 5 days, within 2-3 days, change nutrient solution one time, screening and culturing after 20 days by positive cell clone with 96, 48, 24, 12 and 6 well culture plates amplification culture step by step.Detect the expression amount of GPX1 mutant protein with anti-GPX1 polyclonal antibody and elisa technique, select the cell strain that target protein expression amount is high and support with frozen for spreading cultivation.

3. the expression and purification of people GPX1 mutant protein: the Simultaneous Stabilization that during the 293F that contains 1-10 μ M Sodium Selenite at large capacity spreads cultivation in expressing substratum and supports 2, screening obtains is expressed the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant.In SBP2, SECIS and 293 cells, under the common participation of other protein factor, GPX1 mutant protein is expressed with soluble form and is secreted in substratum, within every 48 hours, collects a nutrient solution.Nutrient solution, after concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, with GSH affinity chromatography column purification GPX1 mutant protein, is collected the elutriant that contains GPX4, and small-molecule substance is removed in dialysis, and freeze-drying obtains people GPX1 mutant protein.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein, its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 7th swimming lane of Fig. 1.Its GPX vigor is 22689U/ μ mol, exceedes order of magnitude of natural GPX.

Embodiment 8: with the target gene of amplification and and the transfection of mammalian cell expression system substep prepare people GPX1 mutant protein

1. the structure of people GPX1 mutant protein expression vector: extract in a small amount test kit (Sigma with mRNA, Cat#MRN-10) from people HepG-2 (DSMZ#ACC180) liver cancer cell, extract mRNA, at reversed transcriptive enzyme (AMV RT, Promega, Cat#M5101) and oligo (dT) exist under, be transcribed into cDNA by RT-PCR (RT-polymerase chain reaction).According to the gene order of disclosed people GPX1 in gene library (referring to NCBI, NM_000581.2) base sequence of encoding gene and 3 ' end non-translational region of design primer amplification GPX1, in the time of design primer, guarantee that 5 of target gene ' end contains initiator codon (ATG) and Hind III restriction enzyme site, the codon of the 2nd Cys replaces with the encoding sequence of Ser, other aminoacid sequence is constant, (from the terminator codon of GPX1 gene, first base is to the previous base of poly oligonucleotide A at whole base sequences of 3 ' end non-translational region of terminator codon downstream introducing GPX1 gene for 3 ' end, concrete sequence is referring to NCBI, and Hind III restriction enzyme site NM_000581.2), there are the target gene of specific restriction enzyme site and cells of mamma animals expression vector (as pSecTag2A etc. with identical sex-limited endonuclease cutting two ends, Invitrogen company), then with DNA ligase by specific restriction enzyme site by GPX1 gene together with its 3 ' end non-translational region be assembled on secretor type cells of mamma animals expression vector, concrete 5 ' end primer is 5 '-CCCAAGCTTCATATGAGTGCTGCTCGGC-3 ', and 3 ' end primer is 5 '-CG GGA TCCAGT GGGGAAACTCG-3 '.Cut latter linked method with first enzyme, by Hind III/BamHI restriction enzyme site, GPX1 gene is connected on the cells of mamma animals expression vector pSecTag2A (Invitrogen company) that same enzyme cuts together with selenocysteine insertion sequence; Keeping under the prerequisite that other aminoacid sequence is constant, according to two rite-directed mutagenesis primers that will sport the halfcystine of Serine and the complete isometric complementation of amino acid whose gene order design near it in GPX1, centered by the codon of mutating acid, the long 25-50bp of primer, with rite-directed mutagenesis primer and Fast Fixed-point sudden change test kit, the sequence encoding mutant of the whole halfcystines in the people GPX1 gene being structured on prokaryotic expression carrier is become to the codon of Serine; Concrete sense primer is respectively: C78S5 '-GTGCTCGGCTTCCCGAGCAACCAGTTTGGGC-3 ', C115S5 '-CATGCTCTTCGAGAAGAGCGAGGTGAACGGTGC-3 ', C156S5 '-CACCTGGTCTCCGGTGAGTCGCAACGATGTTGC-3 ' and C202S5 '-GTCTCAAGGGCCCAGCTCTGCCTAGGGCGCCCCTC-3 '; Determine and suddenly change successfully by DNA sequencing, and occur without other unexpected transgenation, guarantee the gene energy code book invention sequence 1(SEQ ID No:1 after sudden change) described GPX1 mutant protein.According to human selenocysteine insertion sequence in gene library in conjunction with albumen 2(SBP2, NCBI, NM_024077.3) 512 of carboxyl terminal 343-854 position amino acid whose gene orders, its encoding gene of design primer amplification, guarantee that 5 of gene ' end contains initiator codon (ATG) and XbaI enzyme cutting site, 3 ' end contains terminator codon and EcoRI restriction enzyme site; Concrete 5 ' end primer is 5 '-GCTCTAGAATGGAAGCTTTATCTTC-3 ', and 3 ' end primer is 5 '-CGGAATTCTAAATTCAAATTC-3 '.Cut latter linked method with first enzyme, by XbaI/EcoRI restriction enzyme site, the carboxyl terminal of SBP2 (343-854aa) genome is installed in the born of the same parents that same enzyme cuts on type cells of mamma animals expression vector pcDNA3.1/myc-His A (Invitrogen company).

2. the screening of people GPX1 mutant gene positive cell strain: when Growth of Cells to 1 × 10 ⁶when the density of/ml, with the expression vector that contains SBP2 gene at Lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the lower transfection 293F cell (Invitrogen of mediation, R79007), cell was proceeded to and in culture dish, starts adherent culture in after transfection 72 hours, the common 2-50 of adherent culture 48() hour after change the nutrient solution that contains G418, screening has the positive cell clone that G418 resistance is stably express SBP2, G418 concentration is progressively decremented to 200 μ g/ml from 800 μ g/ml during this time, subtract 200 μ g/ml at every turn, each concentration is used 5 days, within 2-3 days, change nutrient solution one time, screening and culturing after 20 days by positive cell clone with 96, 48, 24, 12 and 6 well culture plates amplification culture step by step.Use again the expression vector that contains GPX1 mutant gene at lipofectamine2000 transfection reagent (Invitrogen, by specification uses) the mediation 293F cell (Invitrogen of transfection stably express SBP2 down, R79007), cell was proceeded to and in culture dish, starts adherent culture in after transfection 72 hours, the common 2-50 of adherent culture 48() hour after change the nutrient solution screening that contains G418 and Zeocin to have two kinds of resistances be the positive cell clone that Simultaneous Stabilization is expressed SBP2 and two kinds of foreign proteins of GPX1 mutant, G418 concentration maintains 200 μ g/ml during this time, Zeocin concentration is progressively decremented to 100 μ g/ml from 400 μ g/ml, subtract 100 μ g/ml at every turn, each concentration is used 5 days, within 2-3 days, change nutrient solution one time, screening and culturing after 20 days by the positive cell clone with two kinds of resistances with 96, 48, 24, 12 and 6 well culture plates amplification culture step by step.With the polyclonal antibody of anti-GPX1 and the expression amount of elisa technique detection GPX1 mutant protein, select the cell strain that target protein expression amount is high and support with frozen for spreading cultivation.

Aforesaid method also can first transfection GPX1 mutant gene, rear transfection SBP2 gene.

3. the expression and purification of people GPX1 mutant: the Simultaneous Stabilization that during the 293F that contains 1-10 μ M Sodium Selenite at large capacity spreads cultivation in expressing substratum and supports 2, screening obtains is expressed the cell strain of SBP2 and two kinds of foreign proteins of GPX1 mutant.In SBP2, SECIS and 293F cell, under the common participation of other protein factor, GPX1 mutant is expressed with soluble form and is secreted in substratum, within every 48 hours, collects a nutrient solution.Nutrient solution, after concentrated 20 times of 10KDa molecular weight ultra-filtration membrane, with GSH affinity chromatography column purification GPX1 mutant, is collected the elutriant that contains GPX1 mutant, and small-molecule substance is removed in dialysis, and freeze-drying obtains people GPX1 mutant protein.With denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot) qualification target protein, its molecular weight is 24.2kDa, and is combined with anti-GPX1 antibodies specific, proves successfully to have obtained target protein, sees the 8th swimming lane of Fig. 1.Its GPX vigor is 20968U/ μ mol, exceedes order of magnitude of natural GPX.

Embodiment 9:

Identical with embodiment 1 preparation method, just the encoding sequence of the 87th L-Ala is replaced with when the encoding gene of synthetic GPX1 mutant to the codon (TCC) of Serine, the 87th of the GPX1 mutant protein finally obtaining is Serine, instead of L-Ala, other aminoacid sequence is identical with first method, i.e. sequence 2(SEQ ID No:2 of the present invention) described GPX1 mutant protein.This mutant molecule amount only has small variation, as broad as long in SDS-PAGE and Western Blot result, sees the 9th swimming lane of Fig. 1.But its GPX vigor is 23619U/ μ mol, higher than embodiment 1, exceed order of magnitude of natural GPX.

Embodiment 10:

Identical with embodiment 1 preparation method, just the encoding sequence of the 2nd Serine is replaced with when the encoding gene of synthetic GPX1 mutant to the codon of halfcystine, the second of the GPX1 mutant protein finally obtaining is seleno-cysteine, instead of Serine, other aminoacid sequence is identical with first method, i.e. sequence 3(SEQ ID No:3 of the present invention) described GPX1 mutant protein.This mutant molecule amount only has small variation, as broad as long in SDS-PAGE and Western Blot result, sees the 10th swimming lane of Fig. 1.But its GPX vigor is 15358U/ μ mol, lower than embodiment 1, but still exceed order of magnitude of natural GPX.

Embodiment 11:

Remove expression vector pCold III(TAKARA used embodiment 1, Cat.#3369) change the pCold I(TAKARA with Histidine purification tag into, Cat.#3367), all the other are identical with embodiment 1, obtain the most at last sequence 4(SEQ ID No:4 of the present invention) described GPX1 mutant protein.This mutant has been introduced 16 exogenous amino acids including 6 Histidines on carrier at aminoterminal, therefore molecular weight increases to some extent, in SDS-PAGE and Western Blot result, can see, see the 11st swimming lane of Fig. 1, its advantage is also to use the affine series of strata purification of target of nickel albumen.Its GPX vigor is 20532U/ μ mol, a little less than embodiment 1, proves that purification tag does not have a significant effect to the vigor of albumen, and vigor exceedes order of magnitude of natural GPX.

Claims (4)

1. a Selenoperoxidase GPX1 mutant that contains more than two catalytic group, is characterized in that: its aminoacid sequence is as shown in SEQ ID No:3.

2. a Selenoperoxidase GPX1 mutant that contains more than two catalytic group, is characterized in that: its aminoacid sequence is as shown in SEQ ID No:6.

3. the preparation method of the Selenoperoxidase GPX1 mutant that contains more than two catalytic group claimed in claim 1, its step is as follows:

1), the structure of expression vector:

According to aminoacid sequence SEQ ID No:1, can in auxotrophic strain, express the gene of GPX1 mutant protein with DNA synthesizer synthetic, guarantee that 5 of target gene ' end contains initiator codon ATG, 3 ' end contains terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 49 seleno-cysteine SeCys of target gene replaces with the codon of halfcystine Cys, the encoding sequence of second Serine replaces with the codon of halfcystine, and full length gene does not contain ACA sequence; Then GPX1 mutant gene and the secretor type prokaryotic expression carrier containing specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX1 mutant gene is assembled on secretor type prokaryotic expression carrier with DNA ligase; Described specific restriction enzyme site can be in the multiple clone site of expression vector, contain and non-existent any one restriction enzyme site in GPX1 mutant gene is the DNA sequence dna of the based composition of being identified by restriction endonuclease intrinsic on carrier;

2), the screening of positive transformant and the expression and purification of albumen:

Transform the competent cell of auxotrophic strain-BL21 (DE3) Cys with the expression vector that contains GPX1 mutant gene building in step 1), be coated with the nutrient agar plate containing Cys, screening positive strain; Again the pMazF plasmid that contains express nucleic acid restriction endonuclease MazF is transformed to positive strain, with the M9 solid medium screening positive transformant containing dual anti-property; By positive transformant spread cultivation support after, in the substratum that contains SeCys, essential growth factor and nutrient substance, through 4-25 DEG C of abduction delivering of IPTG low temperature, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX1 mutant that contains SeCys at the combining site of substrate GSH, zymoprotein is expressed and is secreted into soluble form in the pericentral siphon chamber of thalline, and MazF can be by identifying and cut off single stranded RNA particular sequence ACA, suppress the expression of host protein; First cultivate in a small amount the strain of screening high expression level, then enlarged culturing and abduction delivering; Ultrasonication thalline, release zymoprotein under collection, washing, ice bath, by liquid low-temperature centrifugation, remove bacterial sediment, obtain supernatant liquor; With gsh GSH affinitive layer purification GPX1 mutant protein, after dialysis freeze-drying, obtain GPX1 mutant protein sterling, with denaturing polyacrylamide gel electrophoresis SDS-PAGE and Western blot Western blot qualification target protein.

4. the preparation method of the Selenoperoxidase GPX1 mutant that contains more than two catalytic group claimed in claim 2, is characterized in that: be the formation sequence SEQ ID No:6 of institute after the aminoterminal of sequence SEQ ID No:3 is introduced the Histidine purification tag of pColdI prokaryotic expression carrier and the amino acid of factor Xa cleavage site.

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